The multisubunit COP9 signalosome (CSN) complex is found across eukaryotes ranging from fission yeast to humans, and has been implicated in a broad range of biological processes including signal transduction, transcriptional regulation, and plant and animal development. CSN promotes cleavage of the ubiquitin-like protein Nedd8 from cullin proteins, and we propose that this 'deneddylase' activity of CSN underlies its diverse biological functions. Interestingly, a conserved sequence element in the Csn5/Jab1 subunit of CSN that we refer to as the 'Jab1 metalloenzyme motif' (JAMM) is required for the deneddylase activity of CSN. This application proposes to address three major questions that arise from this observation. First, we will employ biochemical and biophysical methods to evaluate the hypothesis that JAMM serves as the active site for a new and conserved class of zinc metalloproteases (Aim 1). Second, by constructing and evaluating the activity of Csn5/Jab1 molecules that harbor point mutations in JAMM, we will test the hypothesis that deneddylase activity underlies one of the best-characterized functions of Csn5/Jab1 by evaluating the ability of mutant Csn5/Jab1 to promote c-Jun-dependent transcription from AP-1 promoter elements (Aim 2). Third, we will seek to determine whether cullins are the exclusive substrates of CSN deneddylase activity, or whether undiscovered substrates may help account for the diverse biological functions of CSN. This objective will be pursued by purification and peptide sequencing of Nedd8-modified proteins that accumulate in CSN-deficient mutants. Given the connections that have been forged between CSN subunits and steroid hormone receptors, cell cycle control proteins, transcriptional regulatory proteins, and cancer-related proteins, a detailed understanding of the the biochemical properties, physiological functions, and physiological targets of CSN's deneddylase activity is likely to shed important light on the biology of normal and diseased human cells.